Molecular interactions, such as protein-protein interactions, are essential components of virtually all cellular processes. The binding of two or more compounds in a cell can have a wide array of effects, including modulating signal transduction, regulating gene transcription, and promoting cellular replication or apoptosis. Several human diseases are associated with malfunctioning of molecular interactions.
Researchers have developed several approaches in attempts to identify molecular interactions. A major breakthrough in the detection of protein-protein interactions was obtained by the introduction of the genetic approaches, of which the yeast two-hybrid (Fields and Song, 1989) is the most important one. Although this technique became widely used, it has several drawbacks. The fusion proteins need to be translocated to the nucleus, which is not always evident. Proteins with intrinsic transcription activation properties may cause false positive signals. Moreover, interactions that are dependent upon secondary modifications of the protein such as phosphorylation cannot be easily detected.
Several alternative systems have been developed to solve one or more of these problems.
Approaches based on phage display do avoid the nuclear translocation. WO9002809 describes how a binding protein can be displayed on the surface of a phage, such as a filamentous phage, wherein the DNA sequence encoding the binding protein is packaged inside the phage. Phages, which bear the binding protein that recognizes the target molecule, are isolated and amplified. Several improvements of the phage display approach have been proposed, as described, e.g., in WO9220791, WO9710330 and WO9732017.
However, all these methods suffer from the difficulties that are inherent to the phage display methodology: the proteins need to be exposed at the phage surface and are so exposed to an environment that is not physiological relevant. Moreover, when screening a phage library, there will be a competition between the phages that results in a selection of the high affinity binders.
A major improvement in the detection of protein-protein interactions was disclosed in WO0190188, describing the so called Mappit system. The method, based on a cytokine receptor, allows not only a reliable detection of protein-protein interactions in mammalian cells, but also modification-dependent protein interactions can be detected, as well as complex three hybrid protein-protein interactions mediated by a small compound (Caligiuri et al., 2006). However, although very useful, the system is limited in sensitivity and some weak interactions cannot be detected. Moreover, as this is a membrane-based system, nuclear interactions are normally not detected. Recently, a cytoplasmic interaction trap has been described, solving several of those shortcomings (WO2012117031). However, all these “genetic” systems rely on the overexpression of both interaction partners, which may result in false positive signals, due to the artificial increase in concentration of the interaction partners.
As an alternative for the genetic protein-protein interaction detection methods described above, biochemical or co-purification strategies combined with mass spectrometry (MS)-based proteomics (Paul et al., 2011; Gingras et al., 2007) can be used. For the co-purification strategies, a cell homogenate is typically prepared by a detergent-based lysis protocol, followed by capture using a (dual) tag approach (Gavin et al., 2002) or via specific antibodies (Malovannaya et al., 2011). The protein complex extracted from the “soup” of cell constituents must then survive several washing steps, mostly to compensate for the sensitivity of contemporary MS instruments, before the actual analysis occurs. There are no clear guidelines on the extent of washing or on available buffers and their stringency. Most lysis and washing protocols are purely empirical in nature and were optimized using model interactions. It is, therefore, hard to speculate on the loss of factors during these steps (false negatives), or the possibility of false interactions due to loss of cellular integrity (false positives). Use of metabolic labeling strategies allows separation between the proteins sticking to the purification matrix, and between the proteins that associate specifically to the bait protein. Depending on the purification conditions and the sensitivity of the MS instruments used, it is no exception to find more than 1000 proteins in the eluted fraction of a gel-free AP-MS experiment (www.crapome.org).
The classical approach to identify target proteins for small molecules relies on the use of “purification handles” that are added to the small molecule. A biotin group is typically used to modify the small molecule, preferentially through a linker and on a permissive site of the molecule. The modified small molecule is then used to capture the associated molecules by a classical pull-down approach using streptavidin beads on a lysate. In a recent implementation, Ong and colleagues describe the use of quantitative proteomics based on metabolic labeling (Stable Isotope Labeling of Amino acids in Cell Culture—SILAC), to define the proteins that bind specifically to a small molecule. The authors use “small-molecule beads” that were prepared by direct chemical coupling of the small molecules to the beads (Ong et al., 2009). Bantscheff and colleagues described a method wherein a panel of broad range kinase inhibitors was coupled to a matrix. This matrix was then incubated with cell lysates to bind a significant portion of the kinome. By adding increasing concentration of candidate kinase inhibitors, on- and off-target kinases can be identified (Bantscheff et al., 2007). A major limitation of this approach is the lack of broad specificity inhibitors outside of the kinase family making it difficult to translate the strategy to other protein target families. In addition, off-targets outside of the kinase family are not readily identified. Another very recent development is thermal profiling to assess the change in thermal stability of proteins upon binding of a small molecule. Proteins tend to aggregate depending on the temperature which is affected by binding of ligands or posttranslational modifications. Savitski and colleagues performed this analysis in a proteome-wide manner using quantitative proteomic approaches and were able to identify known and novel targets for different small molecules (Savitski et al., 2014).
Recently, a co-purification technique has been disclosed in WO2013174999 that allows for evaluating protein-protein interactions in their physiological environment. The complexes are trapped via the p55 GAG protein into artificial virus-like particles (VLPs) that are budded from human cells. The complexes are protected during the enrichment process in a so-called “Virotrap particle.” However, Virotrap, even in its conditional mode of operation, does not identify previously unknown small molecule-protein interactions.
It would be advantageous to entrap small molecule-protein complexes under physiological conditions and thereby evaluate physiologically relevant small molecule-protein interactions.